1. Field of the Invention
This invention relates generally to the use of recombinant DNA methods for genetically altering plants. In particular it relates to the control of ethylene-mediated responses in plants by inhibiting ethylene perception using antisense constructs.
The plant hormone ethylene has a profound influence on plant physiology. Active in trace amounts it affects a number of processes such as fruit ripening, seed germination, plant growth, leaf and flower senescence, pathogen infection, and the interaction of plants with their environment. For instance, ethylene is known to induce a number of physiological changes associated with fruit ripening such as accumulation of carotenoid figments, conversion of cholorplasts to chromoplasts, the increased expression of genes encoding cell wall degradation enzymes fruit softening and susceptibility to pathogens. The importance of ethylene in fruit ripening has been shown by experiments demonstrating that exposure of fruits to specific inhibitors of ethylene biosynthesis greatly retards ripening and that the onset of ripening is hastened when unripe mature green fruits are exposed to exogenous ethylene. Similarly, ethylene is known to promote abscision of leaves, flowers and fruits in a variety of plants species by triggering enzymes that cause cell wall dissolution associated with abscision.
Control of the effects of ethylene is a particularly useful approach to modulating such processes as senescence and fruit ripening. Previous efforts have focused on controlling the amount of ethylene produced by the plant. For example, mutations have been identified that result in production of lower amounts of ethylene during tomato fruit ripening. The fruits from these plants ripen more slowly, and have an extended shelf life. Lack of genetic diversity, however, has frequently limited the success of such breeding programs. Molecular approaches have focused on production of transgenic plants with reduced levels of enzymes required for ethylene biosynthesis. Because plants are extremely sensitive to ethylene, however, it is difficult to completely eliminate the response by reducing ethylene production. Thus, the prior art lacks useful methods for effectively controlling and minimizing the deleterious responses of plants to ethylene.
2. Background Art
The physiological responses of plants to ethylene are reviewed in Liberman, Ann. Rev. Plant Physol. 30:533-591 (1979). The role of ethylene in the regulation of fruit ripening is described in Biale and Young, in Recent Advances in the Biochemistry of the Fruits and Vegetables (Friend and Rhodes, eds, Academic Press, London, 1981).
The cloning of mRNAs that increase in concentration as a result of exposure of tomato fruit to exogenous ethylene is described in Lincoln et al. Proc. Natl. Acad. Sci. U.S.A. 84:2793-2797 (1987). Expression of a gene, designated E8, has been shown to be responsive to ethylene and under strict developmental control (Lincoln and Fischer Mol. Gen. Genet. 212:71-75 (1988) and Lincoln and Fischer Plant Physiol. 88:370-374 (1988). The cDNA sequence of the E8 gene is disclosed in Deikman and Fischer, EMBO J. 7:3315-3320 (1988).
Hiatt et al., U.S. Pat. No. 4,801,340 describes the use if antisense constructs to inhibit polygalacturonase activity in tomato fruit. Transgenic plants have been produced with reduced levels of enzymes required for ethylene biosynthesis Hamilton et al., Nature 346:284 (1990).
Mutants have also been identified which show reduced responsiveness to ethylene. Herner, et al., Plant Physiol. 52:38-42 (1973); Ng, et al., J. Amer. Soc. Hort. Sci. 102:504-509 (1977); Bleecker, et al., Science 241:1086-1089 (1988); and Guzman, et al., Plant Cell 2:513-523 (1990).